Antigenic Characterization of Hantaan and Seoul Virus Nucleocapsid Proteins Expressed by Recombinant Baculovirus: Application of a Truncated Protein, Lacking an Antigenic Region Common to the Two Viruses, as a Serotyping Antigen

Hantaan virus (HTN) and Seoul virus (SEO) are members of the genus Hantavirus in the family Bunyaviridae and are causative agents of hemorrhagic fever with renal syndrome. The complete and truncated nucleocapsid proteins (NP) of HTN and SEO were expressed by a recombinant baculovirus system. Antigenic characterization of the NP using monoclonal antibodies (MAbs) indicated that the binding sites for the serotype-specific MAbs were located between amino acids (aa) 155 and 429. A Western blot assay indicated that the serotype-specific epitopes were conformation dependent. An indirect immunofluorescence antibody (IFA) assay with the truncated NP (aa 155 to 429) was able to distinguish convalescent-phase sera from HTN and SEO patients. However, the antibody titers with the truncated NP were lower than those with the whole NP. The truncated NP of SEO (aa 155 to 429) could be used as an enzyme-linked immunosorbent assay (ELISA) antigen, but the truncated NP from HTN lost its reactivity when used for ELISA. The IFA assay using baculovirus-expressed truncated NP as an antigen is a rapid, simple, and safe test for distinguishing between HTN and SEO infections by serotype.     

Genetic diversities of hantaviruses among rodents in Hokkaido, Japan and Far East Russia

Seroepizootiologic surveys among wild rodents were carried out in Japan and Far East Russia in 1995 and 1996. Seropositive animals were only identified in Clethrionomys rufocanus (23/134) in Hokkaido, Japan. On the other hand, seropositives were identified in C. rufocanus (1/8), Apodemus agrarius (2/66), Apodemus spp. (2/26) and Microtus fortis (3/22) in Vladivostok, Far East Russia. Total RNA was isolated from lungs of seropositive animals and the S genome segments were amplified by PCR, cloned and sequenced. The S and M genomes of hantavirus, derived from Japanese C. rufocanus (Tobetsu genotype), were most closely related with Puumala viruses (76-79% nucleotide and 95% amino acid identities for S genome, 70-78% nucleotide and 87-92% amino acid identities for M genome). The recombinant nucleocapsid protein of Tobetsu genotype was antigenically quite similar with that of Sotkamo. These suggest that the virus endemic in Japanese C. rufocanus belongs to Puumala virus. Phylogenetic analysis indicates that the genotype forms a distinct lineage within Puumala viruses. Partial S segment (1-1251 nt), derived from seropositive M. fortis in Vladivostok, was sequenced and analyzed. The S genome segment, which was designated Vladivostok genotype, was most closely related with Khabarovsk virus (79% nucleotide and 90% amino acid identities) which was isolated from M. fortis.     

A case of food-dependent-exercise induced anaphylaxis possibly induced by shellfish (Sulculus Supertexta and Turbo Cornutus)

A 17 years old girl experienced an anaphylactic reaction of urticaria, dyspnea, syncope and hypotension while riding a bicycle 55 minutes after eating shellfish Lapas shellfish which was a-like Sulculus Supertexta (SS). She recovered within several hours after the emergency treatment. Another attack occurred 3 months later while she was running with a dog 30 minutes after eating shellfish (Turbo Cornutus; TC). RAST scores were 4 for Lapas and 2 for TC. RAST inhibition test by ELISA showed a high crose-reaction between keyhole limpet hemocyanin (KLH) and Lapas, and between KLH and TC, while the cross reaction between Lapas and TC was low. Gel chromatography with sephacryl G-200 revealed that both Lapas and TC had several allergens with different molecules which were detected by ELISA. Exercise challenge produced an immediate fall of FEV1 and a significant increase in plasma histamine levels for 45 minutes.     

α2-Adrenergic receptor subtype alterations in the brainstem in the sudden infant death syndrome

Background: The sudden infant death syndrome (SIDS) is still the main cause of postneonatal infant death. However, the causes and mechanisms of SIDS have never been completely elucidated. Catecholamines, via α2-adrenergic receptor (α2-AR) interactions, are known to influence brainstem autonomic and respiratory activity. Aims: To examine the catecholaminergic system abnormalities in SIDS victims, we investigated the alterations of α2-AR subtypes. Subjects and methods: We examined the developmental changes of α2-AR subtypes in the brainstem, especially in cardiorespiratory nuclei, in 21 SIDS victims and 17 age-matched controls by means of immunohistochemical methods. For statistical analysis, the χ²-test or Fisher’s exact probability test was performed. Results: There was a significant decrease in α2A-AR immunoreactivity in the solitary nucleus and ventrolateral medulla (VLM) in the medulla oblongata in SIDS victims compared with in control cases, but there were no significant differences of the α2B and α2C-AR immunoreactivity in the brainstem between SIDS victims and controls. Conclusion: α2A-AR immunoreactivity was selectively decreased in the solitary nucleus and VLM in the medulla oblongata in SIDS victims, so there was no possibility that it was secondary to chronic hypoxia or repeated ischemia. It may be related to some impairment of the cardiorespiratory neuronal system. Therefore, SIDS victims may be vulnerable to asphyxia, hypoxia, and/or hypercapnia, and fail to exhibit brainstem responses.     

The sensitivity of in situ hybridization for detection of human papillomavirus

We studied a sensitivity of HPV DNA detection by in situ hybridization method using 3H labeled HPV DNA. The materials were CaSki cells and SiHa cells which were derived from as a negative control. The total cellular DNAs extracted from these cell lines were estimated copy numbers of HPV 16 DNA using Southern blot hybridization. In our result, CaSki cell has 400 copies/cell, SiHa cell were appeared to have 1-5 copies/cell. Simultaneously these cells were fixed by periodate-buffered lysine-paraformaldehyde-glutaraldehyde (PLPG) and were detected HPV 16 DNA using in situ hybridization. We detected HPV 16 DNA in CaSki cells and SiHa cells by in situ hybridization also. We concluded that the sensitivity of our in situ hybridization technique is 1-5 copies/cell.     

In vitro antiviral activity of lactoferrin and ribavirin upon hantavirus

Summary.  Bovine lactoferrin (LF) and ribavirin (Rbv) were tested as antiviral agents against Seoul type hantavirus (SR-11 strain) in vitro. Hantaviral foci number in Vero E6 cells infected with SR-11 was reduced with LF treatment by 5 days post infection to obtain a 50% effective dose (ED₅₀) of 2500 μg/ml, while pretreatment with LF was highly efficacious having an ED₅₀ of 39 μg/ml. Conversely, 1 h pretreatment with Rbv revealed no inhibition of viral focus formation but could significantly reduce the number of viral foci (ED₅₀: 10 μg/ml) when used from the time of viral infection. One hour pre-treatment of the cell monolayer with LF and subsequent addition of Rbv revealed a synergistic anti-hantaviral effect against SR-11, <20 FFU/ml as compared to 10⁵ foci/ml in the control. One hour treatment of SR-11 with LF prior to cell inoculation gave an ED₅₀ of 312.5 μg/ml. Whereas, washing the LF-pretreated cell monolayer with PBS demonstrated minimal focus reduction, suggesting LF lightly adheres to cells. These results indicate that LF has anti-hantaviral activity in vitro and inhibition of virus adsorption to cells which play an important role in revealing the anti-hantaviral activity of LF. This paper reports for the first time the anti-hantaviral effect of LF. Received September 21, 1999 Accepted March 15, 2000     

Construction of Canine Herpesvirus Vector Expressing Foreign Genes Using a lacZ-TK Gene Cassette as a Double Selectional Marker

An improved method for constructing canine herpesvirus (CHV) recombinants expressing foreign genes by using the lacZ-TK gene cassette as a double selectional marker was developed. A recombinant CHV carrying the lacZ-TK gene at a targeted gene locus was constructed and used as a parental virus for generating new recombinants. The parental virus formed blue plaques and was sensitive to TK-specific drugs, while newly generated recombinants, in which the lacZ-TK gene was replaced with the desired foreign gene, become both resistant to the TK-specific drugs and formed white plaques. Recombinants were isolated by using the combination of drug selection and color selection. This improved method allows construction of recombinant CHV with great ease, because the drug selection can enrich the frequency of recombinant CHV from 0.01–0.1% to 10–80%. This method was employed to construct a recombinant CHV that expressed rabies virus (RV) glycoprotein (G protein). This revised version was published online in July 2006 with corrections to the Cover Date.